Inhibition of Cxcl4 reduces colony formation in vitro and Cxcl4^−/− HSCs show a reduction in engraftment in secondary BM transplantation assays. (A) WT BM cells enriched for c-Kit⁺ were transduced with a Cxcl4-shRNA vector or control, and mRNA level analyzed by RT-PCR (n = 2). (B-C) Positively transduced cells were selected using GFP, and plated into primary (B) and secondary (C) CFC assays. Data are presented as the mean colony numbers from Cxcl4-shRNA transduced cells or the control. (D) Experimental layout for CD45.2⁺ HSCs from WT or Cxcl4^−/− mice transplanted into irradiated CD45.1⁺ recipients (n = 5 per strain). (E) Engraftment for primary transplant was analyzed in the blood every 4 weeks posttransplant up to 16 weeks. (F) After the primary recipients were sacrificed, CD45.2⁺ LSK cells were transplanted into irradiated recipients (n = 5). Engraftment was analyzed in the blood every 4 weeks posttransplant up to 16 weeks. (G) A percentage of CD45.2⁺ cells was analyzed in different BM populations after 16 weeks from primary transplant, and compared between WT and Cxcl4^−/− mice. (H) Percentage of CD45.2⁺ cells was analyzed in different BM populations after 16 weeks from secondary transplant, and compared between WT and Cxcl4^−/− mice. Data are presented as the mean percentage of CD45.2⁺ cells within the PB. *P < .05; ***P < .001.